The invention relates to the use of a newly identified histone deacetylase inhibitor to facilitate transcription.
The strength of background transcriptional repression, which acts on general-utility promoters, is currently under-appreciated. In some reporter systems, negative effects on transcription (repression) may completely overshadow the positive effects. In other cases, for example, a two-fold induction seen in an experimental situation may not always represent a 100% increase in strength of transactivation per se, but could be mimicked, for example, by a 10% decrease in repression.
It is known that the expression of some genes is regulated by the degree of histone acetylation (Struhl, Genes Dev. 12, 599-606, 1998). Use of a relative nontoxic histone deacetylase (HDAC) inhibitor could, conceivably, simplify the interpretation of transcriptional reporter assays. The use of HDAC inhibition to chemically dissect a pathway should unmask some important measures of pathway activation, which could be overlooked in an undissected system. Indeed, it has been previously observed that the presence of trichostatin A (TSA) or butyrate uncovered the inducibility of certain reporters that initially had appeared inactive (Minucci et al., Proc. Natl. Acad. Sci. U.S.A. 94, 11295-300, 1997; Meng et al., Surgery126, 293-98, 1999).
TSA and butyrate are the most well-studied of the HDAC inhibitors for their effects upon reporters or integrated genes. Various limitations of TSA and butyrate in the applicability to transcriptional assays have been noted in endogenous genes and upon the introduction of exogenous sequences. Butyrate and phenylbutyrate have many functions other than inhibiting HDACs; they have been reported to affect the post-transcriptional modification of other genes (Kitamura et al., Clin. Exp. Immunol. 118, 16-22, 1999) and the depletion of glutamine (Lea and Randolph, Anticancer Res. 18, 2717-22, 1998). There are variable observations that conclude that TSA and other inhibitors do not consistently activate all promoters, and such failures of transcriptional facilitation have included the common general-utility promoters CMV and SV40 (Huang et al., Nature Neurosci. 2, 867-72, 1999; Zhao et al., J. Virol. 73, 5026-33, 1999; Zabel et al., J. Immunol. 163, 2697-703, 1999). Some of the reported transcriptional actions required a specific small recognition element (Li et al., J. Biol. Chem. 274, 7803-15, 1999; Xiao et al., J. Cell. Biochem. 73, 291-302, 1999; Jin and Scotto, Mol. Cell. Biol. 18, 4377-84, 1998), or the activity of a particular co-activator (Sowa et al., Cancer Res. 59, 4266-70, 1999). Furthermore, TSA is not always found to facilitate the detection of positive signal transduction events without interfering with the magnitude of relative transactivation activity (Minucci et al., 1997; Jin et al., 1998). There is, therefore, a need in the art to identify HDAC inhibitors which can be used successfully as general transcription facilitators.
It is an object of the invention to provide reagents and methods of increasing transcription, which can be used for a variety of purposes.
One embodiment of the invention is a pharmaceutical composition comprising 6-(1,3-Dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (scriptaid) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
Another embodiment of the invention is a pharmaceutical composition comprising scriptaid or a pharmaceutically acceptable salt thereof and an expression construct. The expression construct comprises a promoter and a coding sequence for a desired polypeptide. Transcription of the coding sequence is under control of the promoter.
Even another embodiment of the invention is a method of increasing production of a polypeptide. A cell comprising a coding sequence for the polypeptide is contacted with an effective amount of scriptaid or a pharmaceutically acceptable salt thereof. Transcription of the coding sequence is thereby increased relative to transcription in the absence of scriptaid.
Still another embodiment of the invention is a method of increasing production of a polypeptide. A cell-free system comprising a coding sequence for the polypeptide is contacted with an effective amount of scriptaid or a pharmaceutically acceptable salt thereof. Transcription of the coding sequence is thereby increased relative to transcription in the absence of scriptaid.
Yet another embodiment of the invention is a method of increasing production of a therapeutic polypeptide in a diseased cell. The diseased cell is contacted with an effective amount of a composition comprising scriptaid or a pharmaceutically acceptable salt thereof and an expression construct. The expression construct comprises (1) a promoter and (2) a coding sequence for the therapeutic polypeptide. Transcription of the coding sequence is under control of the promoter. Transcription of the coding sequence is thereby increased relative to transcription in the absence of the composition.
Another embodiment of the invention is a kit comprising scriptaid and instructions for a method of increasing production of a polypeptide. The method involves contacting a cell comprising a coding sequence for the polypeptide with an effective amount of scriptaid or a pharmaceutically acceptable salt thereof. Transcription of the coding sequence is thereby increased relative to transcription in the absence of scriptaid.
Even another embodiment of the invention is a method of inhibiting histone deacetylase. The histone deacetylase is contacted with an effective amount of scriptaid or a pharmaceutically acceptable salt thereof. Activity of the histone deacetylase is thereby inhibited relative to activity in the absence of the scriptaid.
Still another embodiment of the invention is a kit comprising scriptaid and instructions for a method of inhibiting histone deacetylase. The method involves contacting the histone deacetylase with an effective amount of scriptaid or a pharmaceutically acceptable salt thereof. Activity of the histone deacetylase is thereby inhibited relative to activity in the absence of the scriptaid.
A further embodiment of the invention is a method of inhibiting histone deacetylase in a neoplastic cell. The neoplastic cell is contacted with an effective amount of scriptaid or a pharmaceutically acceptable salt thereof. Activity of the histone deacetylase is thereby inhibited relative to activity in the absence of the scriptaid.
Even another embodiment of the invention is a method of treating a patient having a tumor. The tumor is contacted with an effective amount of a composition comprising scriptaid or a pharmaceutically acceptable salt thereof. Activity of histone deacetylase in the tumor is thereby inhibited relative to activity in the absence of the scriptaid.
Yet another embodiment of the invention is a method of monitoring expression of a coding sequence in a cell. A cell comprising the coding sequence is contacted with an effective amount of scriptaid or a pharmaceutically acceptable salt thereof. Expression of the coding sequence is thereby increased relative to expression in the absence of the scriptaid. Expression of the coding sequence is assayed.
The invention thus provides reagents and methods for increasing production of a desired polypeptide, for inhibiting histone deacetylase, and monitoring expression of a polypeptide, as well as therapeutic methods for treating patients with tumors.